ABSTRACT One key goal of the NCI Precision Medicine Initiative focuses on development of new tools to tailor cancer therapy to disease status and risk of metastasis-- Patients at high risk for metastatic disease would receive aggressive, frequently molecularly-targeted therapy, whereas those with low risk for metastatic disease would be treated with appropriate local therapies, sparing them toxic side effects of therapy while maintaining high likelihood for cure. One of the main challenges preventing implementation of precision medicine for metastasis is limited understanding of signaling molecules and pathways that confer high metastatic potential to a small subset of cancer cells within a larger, heterogeneous tumor. Consequently, molecular imaging of metastatic ?potential? is an unvanquished challenge. To engineer biosensors that can detect and measure metastatic 'potential' of single living cancer cells, we carried out a comprehensive analysis of the pan-cancer phosphoproteome to search for actin-remodelers required for cell migration, that are enriched in cancers, but excluded in normal cells. Only one phosphoprotein emerged, tyr-phosphorylated CCDC88A (GIV/Girdin), a bona-fide metastasis-related protein across a variety of solid tumors. We designed multi-modular biosensors that are partly derived from GIV, and because GIV integrates pro-metastatic signaling by multiple oncogenic receptors, we named them ?Integrator-of-Metastatic- Potential (IMP)'. It is hypothesized that single cell imaging of GIV activation using IMPs, rather than simply levels of GIV expression, will detect the subset of metastasis-initiating cells that must be eliminated to prevent metastatic disease. Preliminary experiments demonstrate that IMPs captured the heterogeneity of metastatic potential within primary lung and breast tumors at steady-state, detected those few cells which have acquired the highest metastatic potential and tracked their enrichment during metastasis. These findings provide proof- of-concept that IMPs can measure the diversity and plasticity of metastatic potential of tumor cells in a sensitive and unbiased way. Going forward, IMPs will be optimized and validated for use as tools for molecular imaging of metastasis-initiating cells via 3 goals: 1) Image metastatic potential of single breast cancer cells; 2) Image GIV activation as a marker of Metastasis Initiating Cells (MICs); and 3) Image metastatic potential in patient- derived xenografts. To accomplish these goals, this multi PD/PI proposal capitalizes on synergy of non- overlapping expertise of two PIs and two animal model systems (zebrafish and mice), in-depth biology of a novel signaling pathway, and cutting-edge technology of in vivo imaging. Success in detecting metastasis-initiating cells will be a transformative advance for cancer cell biology. It will pave the path for the development of personalized screening platforms to assess the effectiveness of anti- cancer drugs in killing the highly tumorigenic cells within any given tumor, and ultimately enable clinical implementation of precision cancer therapy to improve treatment and quality of life for patients.